U.S. patent number 6,068,850 [Application Number 09/248,286] was granted by the patent office on 2000-05-30 for aqueous formulations of peptides.
This patent grant is currently assigned to Alza Corporation. Invention is credited to James B. Eckenhoff, deceased, John J. Leonard, Jr., Steven J. Prestrelski, Cynthia L. Stevenson, Sally A. Tao, Jeremy C. Wright.
United States Patent |
6,068,850 |
Stevenson , et al. |
May 30, 2000 |
Aqueous formulations of peptides
Abstract
This invention relates to stable liquid aqueous formulations of
peptide compounds at high concentrations. These stable formulations
comprise at least about 10% peptide in water. They may be stored at
elevated temperatures for long periods of time and are especially
useful in implantable delivery devices for long term delivery of
drug.
Inventors: |
Stevenson; Cynthia L. (Mountain
View, CA), Tao; Sally A. (San Jose, CA), Prestrelski;
Steven J. (Mountain View, CA), Eckenhoff, deceased; James
B. (late of Los Altos, CA), Wright; Jeremy C. (Los
Altos, CA), Leonard, Jr.; John J. (Cupertino, CA) |
Assignee: |
Alza Corporation (CA)
|
Family
ID: |
21802912 |
Appl.
No.: |
09/248,286 |
Filed: |
February 11, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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874694 |
Jun 13, 1997 |
5916582 |
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Current U.S.
Class: |
424/423 |
Current CPC
Class: |
A61K
38/09 (20130101); A61K 9/0024 (20130101); A61P
35/00 (20180101); A61P 43/00 (20180101); A61P
31/00 (20180101); A61K 9/08 (20130101); A61K
9/0019 (20130101); A61P 13/08 (20180101); A61P
15/00 (20180101); A61K 47/20 (20130101) |
Current International
Class: |
A61K
38/08 (20060101); A61K 38/09 (20060101); A61K
47/20 (20060101); A61K 9/08 (20060101); A61K
9/00 (20060101); A61F 002/02 () |
Field of
Search: |
;424/423 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0111841 |
|
Jun 1984 |
|
EP |
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WO92/20711 |
|
Nov 1992 |
|
WO |
|
WO94/19020 |
|
Sep 1994 |
|
WO |
|
WO95/01183 |
|
Jan 1995 |
|
WO |
|
WO95/00168 |
|
Jan 1995 |
|
WO |
|
WO95/04540 |
|
Feb 1995 |
|
WO |
|
WO96/07398 |
|
Mar 1996 |
|
WO |
|
WO97/27840 |
|
Aug 1997 |
|
WO |
|
Other References
Fu Lu, et al., "Percutaneous Absorption Enhancement of Leuprolide",
Pharm. Res., 9(12):1575-1576 (1992). .
Helm, et al., "Stability of Gonadorelin and Triptorelin in Aqueous
Solution", Pharm. Res., 7(12):1253-1256 (1990). .
Johnson, et al., "Degradation of the LH-RH Analog Nafarelin Acetate
in Aqueous Solution", Intl. J. Pharm., 31:125-129 (1986). .
Okada, et al., "Preparation of Three-Month Depot Injectable
Microspheres of Leuprorelin Acetate Using Biodegradable Polymers",
Pharm. Res., 11(8):1143-1147 (1994). .
Okada, et al., "New Degradation Product of Des-Gly.sup.10 -NH.sub.2
-LH-RH-Ethylamide (Fertirelin) in Aqueous Solution", J. Pharm.
Sci., 80(2):167-170 (1991). .
Oyler, et al., "Characterization of the Solution Degradation
Products of .
Histrelin, a Gonadotropin Releasing Hormone (LH/RH) Agonist", J.
Pharm. Sci., 80(3):271-275 (1991). .
Powell, et al., "Peptide Liquid Crystals: Inverse Correlation of
Kinetic Formation and Thermodynamic Stability in Aqueous Solution",
Pharm. Res., 11(9):1352-1354 (1994). .
Powell, et al., "Parenteral Peptide Formulations: Chemical and
Physical Properties of Native Luteinizing Hormone-Releasing Hormone
(LHRH) and Hydrophobic Analogues in Aqueous Solution", Pharm. Res.,
8(10):1258-1263 (1991). .
Powers, et al., "Solution Behavior of Leuprolide Acetate, an LHRH
Agonist, as Determined by Circular Dichroism Spectroscopy", Intl.
J. Pharm.,108:49-55 (1994). .
Shi, et al., "Long-Term Stability of Aqueous Solutions of
Luteinizing Hormone-Releasing Hormone Assessed by an In-Vitro
Bioassay and Liquid Chromatography", J. Pharm. Sci., 73(6):819-821
(1984). .
Toguchi, "Pharmaceutical Manipulation of Leuprorelin Acetate to
Improve Clinical Performance", J. Intl. Med. Res., 18:35-41 (1990).
.
Factrel (gonadorelin HCI for subcutaneous or IV injection),
Physician's Desk Reference, 50th Edition, pp. 2877-2878 (1996).
.
Lupron (leuprolide acetate for subcutaneous injection), Physician's
Desk Reference, 50th Edition, pp. 2555-2556 (1996). .
Lupron Depot (leuprolide acetate for depot suspension), Physician's
Desk Reference, 50th Edition, pp. 2556-2562 (1996). .
Lutrepulse (gonadorelin acetate for IV injection), Physician's Desk
Reference, 50th Edition, pp. 980-982 (1996). .
Zoladex (goserelin acetate implant), Physician's Desk Reference,
50th Edition, pp. 2858-2861 (1996)..
|
Primary Examiner: Azpuru; Carlos A
Attorney, Agent or Firm: Dillahunty; Mary Ann Stone; Steven
F. Clarke; Pauline A.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of application Ser. No.
08/874,694, filed Jun. 13, 1997, now U.S. Pat. No. 5,916,582, which
is a continuation-in-part of application No. 60/021,199 filed Jul.
3, 1996.
Claims
What is claimed is:
1. A stable aqueous formulation of a peptide related compound
comprising:
a) at least about 10% (w/w) of at least one peptide compound;
and
b) water, wherein said formulation is stable at 37.degree. C. for
at least 2 months.
2. The formulation of claim 1 which comprises at least about 30%
(w/w) peptide compound.
3. The formulation of claim 1 wherein said peptide compound is an
LHRH-related compound.
4. The formulation of claim 3 wherein said peptide compound is
selected from the group consisting of leuprolide, LHRH, nafarelin
and goserelin.
5. The formulation of claim 1 which is stable after
irradiation.
6. The formulation of claim 1 which is stable at 37.degree. C. for
at least one year.
7. The formulation of claim 1 which is adapted for use in an
implantable drug delivery device.
8. The formulation of claim 1 which further comprises at least one
selected from the group consisting of a buffer, an excipient, a
solvent, a solubilizer and a preservative.
9. The formulation of claim 1 which consists essentially of about
30% to about 50% (w/w) of the LHRH-related compound leuprolide
acetate in sterile distilled water.
10. The formulation of claim 1 which forms a gel.
11. The formulation of claim 1 further comprising at least one
non-aqueous polar aprotic solvent.
12. The formulation of claim 11 wherein said non-aqueous polar
aprotic solvent is DMSO or DMF.
13. A method for preparing the stable aqueous formulation of claim
1 comprising dissolving at least about 10% (w/w) of at least one
peptide compound in water.
14. The method of claim 13 wherein at least about 30% (w/w) peptide
compound is dissolved.
15. The method of claim 13 wherein said peptide compound is an
LHRH-related compound.
16. The method of claim 15 wherein said peptide compound is
selected from the group consisting of leuprolide, LHRH, nafarelin
and goserelin.
17. The method of claim 13 further comprising the step of adding at
least one selected from the group consisting of a buffer, an
excipient, a solvent, a solubilizer and a preservative.
18. The method of claim 13 wherein about 30% to about 50% (w/w) of
the LHRH-related compound leuprolide acetate is dissolved in
sterile distilled water.
19. The method of claim 13 further comprising the step of adding at
least one non-aqueous polar aprotic solvent.
20. The method of claim 19 wherein said non-aqueous polar aprotic
solvent is DMSO or DMF.
21. A method for treating a subject suffering from a condition
which may be alleviated by administration of an peptide compound
comprising administering to said subject an effective amount of the
formulation of claim 1.
22. The method of claim 21 wherein said administration is
parenteral administration.
23. The method of claim 21 wherein said administration is long-term
continuous administration.
24. The method of claim 23 wherein said administration is
accomplished by use of an implantable drug delivery device.
25. The method of claim 21 wherein said condition is prostatic
cancer and said peptide compound is leuprolide.
26. The method of claim 21 wherein at least about 80 micrograms of
leuprolide is administered daily.
27. The method of claim 26 wherein said daily administration
continues for a period selected from the group consisting of 3
months, 6 months and 12 months.
28. The method of claim 27 wherein said daily administration for
said period is continuous administration accomplished using an
implantable drug delivery system.
29. The method of claim 21 wherein said condition is prostatic
cancer and said peptide compound is an LHRH antagonist.
30. The formulation of claim 1 which is stable at 37.degree. C. for
at least 3 months.
Description
FIELD OF THE INVENTION
This invention relates to stable aqueous formulations of peptide
compounds at high concentrations.
BACKGROUND OF THE INVENTION
References
The following references are referred to by numbers in brackets ([
]) at the relevant portion of the specification.
1. Zoladex (goserelin acetate implant), Physician's Desk Reference,
50th Edition, pages 2858-2861 (1996).
2. U.S. Pat. No. 3,914,412, issued Oct. 21, 1975.
3. U.S. Pat. No. 4,547,370, issued Oct. 15, 1985.
4. U.S. Pat. No. 4,661,472, issued Apr. 28, 1987.
5. U.S. Pat. No. 4,689,396, issued Aug. 25, 1987.
6. U.S. Pat. No. 4,851,385, issued Jul. 25, 1989.
7. U.S. Pat. No. 5,198,533, issued Mar. 30, 1993.
8. U.S. Pat. No. 5,480,868, issued Jan. 2, 1996.
9. WO92/20711, published Nov. 26, 1992.
10. WO95/00168, published Jan. 5, 1995.
11. WO95/04540, published Feb. 16, 1995.
12. "Stability of Gonadorelin and Triptorelin in Aqueous Solution",
V. J. Helm, B. W. Muller, Pharmaceutical Research, 7/12, pages
1253-1256 (1990).
13. "New Degradation Product of Des-Gly.sup.10 --NH.sub.2
--LH--RH-Ethylamide (Fertirelin) in Aqueous Solution", J. Okada, T.
Seo, F. Kasahara, K. Takeda, S. Kondo, J. of Pharmaceutical
Sciences, 8012, pages 167-170 (1991).
14. "Characterization of the Solution Degradation Product of
Histrelin, a Gonadotropin Releasing Hormone (LHRH) Agonist", A. R.
Oyler, R. E. Naldi, J. R. Lloyd, D. A. Graden, C. J. Shaw, M. L.
Cotter, J. of Pharmaceutical Sciences, 80/3, pages 271-275
(1991).
15. "Parenteral Peptide Formulations: Chemical and Physical
Properties of Native Luteinizing Hormone-Releasing Hormone (LHRH)
and Hydrophobic Analogues in Aqueous Solution", M. F. Powell, L. M.
Sanders, A. Rogerson, V. Si, Pharmaceutical Research, 8/10, pages
1258-1263 (1991).
16. "Degradation of the LHRH Analog Nafarelin Acetate in Aqueous
Solution", D. M. Johnson, R. A. Pritchard, W. F. Taylor, D. Conley,
G. Zuniga, K. G. McGreevy, Intl. J. of Pharmaceutics, 31, pages
125-129 (1986).
17. "Percutaneous Absorption Enhancement of Leuprolide", M. Y. Fu
Lu, D. Lee, G. S. Rao, Pharmaceutical Research, 9/12, pages
1575-1576 (1992).
18. Lutrepulse (gonadorelin acetate for IV injection), Physician's
Desk Reference, 50th Edition, pages 980-982 (1996).
19. Factrel (gonadorelin HCl for subcutaneous or IV injection),
Physician's Desk Reference, 50th Edition, pages 2877-2878
(1996).
20. Lupron (leuprolide acetate for subcutaneous injection),
Physician's Desk Reference, 50th Edition, pages 2555-2556
(1996).
21. Lupron depot (leuprolide acetate for depot suspension),
Physician's Desk Reference, 50th Edition, pages 2556-2562
(1996).
22. "Pharmaceutical Manipulation of Leuprorelin Acetate to Improve
Clinical Performance", H. Toguchi, J. of Intl. Medical Research,
18, pages 35-41 (1990).
23. "Long-Term Stability of Aqueous Solutions of Luteinizing
Hormone-Releasing Hormone Assessed by an In-Vitro Bioassay and
Liquid Chromatography", Y. F. Shi, R. J. Sherins, D. Brightwell, J.
F. Gallelli, D. C. Chatterji, J. of Pharmaceutical Sciences, 73/6,
pages 819-821 (1984).
24. "Peptide Liquid Crystals: Inverse Correlation of Kinetic
Formation and Thermodynamic Stability in Aqueous Solution", M. F.
Powell, J. Fleitman, L. M. Sanders, V. C. Si, Pharmaceutical
Research, 11/9, pages 1352-1354 (1994).
25. "Solution Behavior of Leuprolide Acetate, an LHRH Agonist, as
Determined by Circular Dichroism Spectroscopy", M. E. Powers, A.
Adejei, M. Y. Fu Lu, M. C. Manning, Intl. J. of Pharmaceutics, 108,
pages 49-55 (1994).
26. "Preparation of Three-Month Depot Injectable Microspheres of
Leuprorelin Acetate Using Biodegradable Polymers", Pharmaceutical
Research, 1118, pages 1143-1147 (1994).
The disclosure of each of the above publications, patents or patent
applications is hereby incorporated by reference in its entirety to
the same extent as if the language of each individual publication,
patent and patent application were specifically and individually
incorporated by reference.
Luteinizing hormone-releasing hormone (LHRH), also known as
gonadotropin releasing hormone (GnRH), is a decapeptide with the
structure:
It is secreted by the hypothalamus and binds to receptors on the
pituitary gland, releasing luteinizing hormone (LH) and follicle
stimulating hormone (FSH). LH and FSH stimulate the gonads to
synthesize steroid hormones. Numerous analogs of LHRH are known,
including peptides related to LHRH which act as agonists and those
which act as antagonists. [1-15] LHRH analogs are known to be
useful for treating hormone-dependent diseases such as prostate
cancer, benign prostatomegaly, endometriosis, hysteromyoma,
metrofibroma, precocious puberty, or mammary cancer and as
contraceptives. [8] Sustained release administration is preferred
for both agonist LHRH-related compounds, which reduce the number of
available receptors after repeated administration so that the
production of steroid hormones is suppressed, and antagonist
LHRH-related compounds, which must be continually administered for
persistent inhibition of endogenous LHRH. [8]
The sustained parenteral delivery of drugs, especially peptide
drugs, provides many advantages. The use of implantable devices for
sustained delivery of a wide variety of drugs or other beneficial
agents is well known in the art. Typical devices are described, for
example, in U.S. Pat. Nos. 5,034,229; 5,057,318; and 5,110,596. The
disclosure of each of these patents is incorporated herein by
reference.
In general, oral bioavailability of peptides, including
LHRH-related compounds, is low. [16-17]
Currently marketed aqueous formulations of LHRH, its analogs and
related compounds which are used for parenteral injection generally
contain relatively low concentrations of LHRH-related compounds
(0.05 to 5 mg/ml) and may also contain excipients such as mannitol
or lactose. [18-20] Such formulations of LHRH-related compounds
must either be stored refrigerated or may be stored at room
temperature for short periods of time.
Available depot formulations of LHRH-related compounds administered
for sustained release over a period of 1-3 months include a
formulation comprised of 15% LHRH-related compound dispersed in a
matrix of D,L-lactic and glycolic acids copolymer presented as a
cylinder to be injected subcutaneously [1] and a formulation
comprised of microparticles comprising a core of LHRH-related
compound and gelatin surrounded by a shell of D,L-lactic and
glycolic acids copolymer. These microparticles are suspended in a
diluent for injection either subcutaneously or intramuscularly.
[21, 26] These products must be stored at room temperature or
lower. Aqueous formulations of LHRH-related compounds are known to
exhibit both chemical and physical instability, as well as
degradation after irradiation. [12-16, 22-25]
Formulations which have been shown to be stable (t.sub.90 about
five years) have been very low concentration (25 .mu.g/ml) aqueous,
buffered (10 mM buffer, ionic strength of 0.15) solutions stored at
temperatures no higher than room temperature (25.degree. C.).
[15]
There is a need for stable, high concentration aqueous formulations
of peptides.
SUMMARY OF THE INVENTION
The present invention provides stable aqueous formulations which
are solutions of peptides compounds in water at concentrations of
at least about 10%. These stable high concentration formulations
may be stored at elevated temperatures (e.g., 37.degree. C.) for
long periods of time and are especially useful in implantable
delivery devices for long term delivery (e.g., 1-12 months or
longer) of drug. The aqueous formulations may optionally include
buffer, excipients, ethanol (EtOH), a surfactant or a
preservative.
In one aspect, the invention provides stable aqueous formulations
of peptide compounds, said formulations comprising at least about
10% (w/w) peptide compound and water.
In another aspect, the invention provides methods for preparing a
stable aqueous formulation of an peptide compound, said methods
comprising dissolving at least about 10% (w/w) peptide compound in
water.
In yet a further aspect, the invention provides methods for
treating a subject suffering from a condition which may be
alleviated by administration of a peptide compound, said methods
comprising administering to said subject an effective amount of a
stable aqueous formulation comprising at least about 10% (w/w)
peptide compound and water .
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the stability of 40% leuprolide acetate solution
in water after two months at 80.degree. C. as measured by reverse
phase HPLC (RP-HPLC).
FIG. 2 shows the same sample as FIG. 1 injected by size exclusion
chromatography (SEC). This figure shows that there is very little
aggregation, and what aggregation there is is comprised of dimer
and trimer products, with no higher order aggregation.
FIG. 3 presents the Arrhenius plot showing the loss of leuprolide
from 40% solutions of leuprolide acetate in water.
FIG. 4 illustrates the chemical and physical stability of a 40%
leuprolide acetate solution in water after about three months at
80.degree. C.
FIG. 5 illustrates the loss of leuprolide acetate fit to pseudo
first order kinetics from a 40% solution in water over a period of
three to six months at 37.degree. C., 50.degree. C., 65.degree. C.
and 80.degree. C.
FIG. 6 illustrates the chemical and physical stability of a 40%
leuprolide acetate solution in water after nine months at
37.degree. C.
FIG. 7 illustrates the stability of a 30% goserelin solution in
acetate buffer and mannitol after 14 days at 80.degree. C.
FIG. 8 illustrates that both gelled and non-gelled aqueous
formulations of leuprolide (370 mg/ml) remained stable over a
period of 6 months at 37.degree. C.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is drawn to the unexpected discovery that
dissolving high concentrations (i.e., at least about 10%) of
peptide compounds in water results in stable aqueous formulations.
Previously known aqueous formulations of peptide compounds, which
are dilute buffered aqueous solutions containing excipients such as
EDTA or ascorbic acid which must be stored at low temperatures
(4-25.degree. C.), form degradation products using degradation
pathways such as acid/base catalyzed hydrolysis, deamidation,
racemization and oxidation. In contrast, the presently claimed
formulations stabilize peptide compounds at high concentrations at
elevated temperatures (e.g., 37.degree. C. to 80.degree. C.), thus
making possible the delivery of peptides in implantable delivery
devices that would not otherwise be feasible.
Standard peptide and protein formulations consist of dilute aqueous
solutions. Two critical aspects of peptide formulation include
solubilization and stabilization of the drug molecule. Peptide
stability is usually achieved by varying one or more of the
following: pH, buffer type, ionic strength, excipients (EDTA,
ascorbic acid, etc.). In contrast, in the present invention, highly
concentrated peptides formulated in water provide stable
solutions.
The invention consists of using high concentrations of peptide in
aqueous solution to stabilize the peptide formulations against both
chemical and physical degradation.
A. Definitions
As used herein, the following terms have the following
meanings:
The term "chemical stability" means that an acceptable percentage
of degradation products produced by chemical pathways such as
oxidation or hydrolysis is formed. In particular, a formulation is
considered chemically stable if no more than about 20% breakdown
products are formed after two months at 37.degree. C.
The term "physical stability" means that an acceptable percentage
of aggregates (e.g., dimers, trimers and larger forms) is formed.
In particular, a formulation is considered physically stable if no
more that about 15% aggregates are formed after two months at
37.degree. C.
The term "stable formulation" means that at least about 65%
chemically and physically stable peptide compound remains after two
months at 37.degree. C. (or equivalent conditions at an elevated
temperature). Particularly preferred formulations are those which
retain at least about 80% chemically and physically stable peptide
under these conditions. Especially preferred stable formulations
are those which do not exhibit degradation after sterilizing
irradiation (e.g., gamma, beta or electron beam).
The terms "peptide" and/or "peptide compound" mean polymers of up
to about 50 amino acid residues bound together by amide (CONH)
linkages. Analogs,
derivatives, agonists, antagonists and pharmaceutically acceptable
salts of any of these are included in these terms. The terms also
include peptides and/or peptide compounds which have D-amino acids,
modified, derivatized or non-naturally occurring amino acids in
their D- or L-configuration and/or peptomimetic units as part of
their structure.
The term "LHRH-related compound" means luteinizing hormone
releasing hormone (LHRH) and its analogs and pharmaceutically
acceptable salts. Octa-, nona- and decapeptide LHRH agonists and
antagonists are included in the term LHRH-related compounds, as is
native LHRH. Particularly preferred LHRH-related compounds include
LHRH, leuprolide, goserelin, nafarelin, and other known active
agonists and antagonists. [1-21]
The term "high concentration" means at least about 10% (w/w) and up
to the maximum solubility of the particular LHRH-related
compound.
The term "excipient" means a more or less inert substance in a
formulation which is added as a diluent or vehicle or to give form
or consistency. Excipients are distinguished from solvents such as
EtOH, which are used to dissolve drugs in formulations, from
non-ionic surfactants such as Tween 20, which are used to
solubilize drugs in formulations, and from preservatives such as
benzyl alcohols or methyl or propyl parabens, which are used to
prevent or inhibit microbial growth.
The term "buffering capacity" means the capacity of a solution due
to the presence of a mixture of an acid/base pair in the solution
to reduce any changes in pH that would otherwise occur in the
solution when acid or alkali is added to it.
The term "polar aprotic solvent" means a polar solvent which does
not contain acidic hydrogen and does not act as a hydrogen bond
donor. Examples of polar aprotic solvents are dimethylsulfoxide
(DMSO), dimethylformamide (DMF), hexamethylphosphorotriamide
(HMPT), and n-methyl pyrrolidone.
B. Preparation of Formulations
The present invention is drawn to highly concentrated liquid
aqueous formulations of peptide compounds which are stable for
prolonged periods of time at elevated temperatures. Standard dilute
aqueous peptide and protein formulations require manipulation of
buffer type, ionic strength, pH and excipients (e.g., EDTA and
ascorbic acid) to achieve stability. In contrast, the claimed
formulations achieve stabilization of peptide compounds by the use
of high concentrations (at least about 10%, w/w) of compound
dissolved in water.
Examples of peptides and peptide compounds which may be formulated
using the present invention include those peptides which have
biological activity or which may be used to treat a disease or
other pathological condition. They include, but are not limited to
adrenocorticotropic hormone, angiotensin I and II, atrial
natriuretic peptide, bombesin, bradykinin, calcitonin, cerebellin,
dynorphin A, alpha and beta endorphin, endothelin, enkephalin,
epidermal growth factor, fertirelin, follicular gonadotropin
releasing peptide, galanin, glucagon, gonadorelin, gonadotropin,
goserelin, growth hormone releasing peptide, histrelin, insulin,
leuprolide, LHRH, motilin, nafarelin, neurotensin, oxytocin,
somatostatin, substance P, tumor necrosis factor, triptorelin, and
vasopressin. Analogs, derivatives, antagonists, agonists and
pharmaceutically acceptable salts of the above may also be
used.
Depending on the particular peptide compound to be formulated,
ionic strength and pH may be factors worthy of consideration. For
example, we have found that preferred aqueous formulations of
leuprolide acetate have low ionic strength and pH between about 4
and about 6.
The peptide compounds useful in the formulations and methods of the
present invention can be used in the form of a salt, preferably a
pharmaceutically acceptable salt. Useful salts are known to those
of skill in the art and include salts with inorganic acids, organic
acids, inorganic bases or organic bases. Preferred salts are
acetate salts.
Peptide compounds which are hydrophilic and readily soluble in
water are preferred for use in the present invention. One of skill
in the art can easily determine which compounds will be useful on
the basis of their aqueous solubility, i.e., the compound must be
soluble in water to at least about 10% (w/w). Preferably, this is
also a pharmaceutically effective amount. Particularly preferred
peptide compounds are LHRH-related compounds, including leuprolide
and leuprolide acetate.
The proportion of peptide may vary depending on the compound, the
condition to be treated, the solubility of the compound, the
expected dose and the duration of administration. (See, for
example, The Pharmacological Basis of Therapeutics, Gilman et al.,
7th ed. (1985) and Pharmaceutical Sciences, Remington, 18th ed.
(1990), the disclosures of which are incorporated herein by
reference.) The concentration of peptide compound may range from at
least about 10% (w/w) to the maximum solubility of the compound. A
preferred range is from about 20 to about 60% (w/w). The currently
more preferred range is from about 30 to about 50% (w/w) and a most
preferred range is about 35 to about 45% (w/w).
Generally, the stable formulations of the present invention may be
prepared by simply dissolving a therapeutically effective amount of
the desired peptide compound in water, although pH adjustments may
be made.
It is known to those of skill in the art that buffers, excipients,
solvents such as EtOH, solubilizers such as non-ionic surfactants,
and preservatives may beneficially be added to pharmaceutical
peptide formulations. (See, for example, Pharmaceutical Sciences,
Remington, 18th ed. (1990).) Such agents may optionally be added to
the claimed formulations.
C. Methodology
We have found that stable aqueous formulations of peptide compounds
may be prepared by dissolving a high concentration (at least about
10%) of the peptide compound to be formulated in water.
We have tested these peptide compound formulations, specifically
formulations of the LHRH-related compound leuprolide, for stability
by subjecting them to accelerated aging at elevated temperature and
measuring the chemical and physical stability of the formulations.
Results of these studies (shown, for example, in Table III and
FIGS. 1, 2 and 6) demonstrate that these formulations were stable
at conditions that approximate or exceed storage for one year at
37.degree. C.
We have also tested peptide compound formulations prepared as
described herein for stability after 2.5 megarad gamma irradiation.
Results, shown in Table IV, show that these formulations remained
chemically and physically stable after such irradiation.
Formulations subjected to electron beam irradiation were also found
to be stable.
As shown in Table I, we have tested a wide variety of peptide
formulations, specifically leuprolide, goserelin, LHRH, angiotensin
I, bradykinin, calcitonin, insulin, trypsinogen and vasopressin,
for stability by dissolving (or attempting to dissolve) them in
water, then subjecting them to accelerated aging at elevated
temperatures. The stability of the formulations was measured.
Results are presented in Table I as half-life at 37.degree. C.
assuming an E.sub.a =22.2 kcal/mole. A wide range of the peptides
tested were soluble in water and remained stable under the test
conditions. The solubility of a particular peptide in water and the
stability of the resulting solution are easily determined using
routine procedures known to those of ordinary skill in the art.
TABLE I ______________________________________ Stability of
Peptides Formulated in Water HALF-LIFE* FORMULATION (Temperature)
______________________________________ 40% Leuprolide 9.7 years
(37.degree. C.) 40% Goserelin 19.3 months (80.degree. C.) 20% LHRH
2.5 years (65.degree. C.) 20% Angiotensin I insoluble gel
(65.degree. C.) 20% Bradykinin 8.5 months (65.degree. C.) 40%
Calcitonin insoluble (80.degree. C.) 20% Calcitonin 9.6 months
(80.degree. C.) 5% Calcitonin 23.5 months (50.degree. C.) 20%
Insulin insoluble gel (65.degree. C.) 40% Trypsinogen insoluble gel
(65.degree. C./80.degree. C.) 20% Trypsinogen insoluble gel
(65.degree. C.) 40% Vasopressin degraded (80.degree. C.) 20%
Vasopressin 14.3 days (65.degree. C.) *Half-life at 37.degree. C.
assuming E.sub.a = 22.2 kcal/mole.
______________________________________
Formulations of 40% leuprolide in water stored for six months at
37.degree. C. showed linear degradation as measured by overall loss
of peptide from the solution. Analysis of these data gave an
activation energy (E.sub.a) of 22.2 kcal/mole and a t.sub.90 of
13.8 months, showing stability of these formulations at elevated
temperatures.
We have also unexpectedly found that certain peptide formulations
of the present invention are bacteriostatic (i.e., inhibit
bacterial growth), bactericidal (i.e., cause the death of
bacteria), and sporicidal (i.e., kill spores). In particular,
leuprolide formulations of 50-400 mg/ml exhibited bacteriostatic,
bactericidal and sporicidal activity. The stability of the samples
was unaffected by spiking with bacteria, indicating that the
enzymes released from the killed and lysed bacteria did not
adversely affect the stability of the product. This demonstrates
that these formulations were not conducive to enzymatic
activity.
Some peptides, for example calcitonin and leuprolide, are known to
be physically unstable, exhibiting aggregation, gelation and
fibrillation when formulated in aqueous solution. For example,
leuprolide can be induced to gel by increasing peptide
concentration, introduction of salts or gentle agitation. Improving
physical stability can allow for easier parenteral administration,
including administration using implantable drug delivery
systems.
It has unexpectedly been found that adding polar aprotic solvents
such as DMSO to aqueous formulations of certain peptides, such as
leuprolide, goserelin and calcitonin, prevents gelation of the
formulation. This is apparently because non-aqueous polar aprotic
solvents cause peptides to form a random coil/alpha helix
conformation that does not refold into a beta sheet structure and,
therefore, does not gel. Thus, these solvents have an anti-gellant
effect.
Additionally, studies of gelled and non-gelled aqueous formulations
of leuprolide (370 mg/ml) stored at 37.degree. C. for 6 weeks
showed a similar chemical stability profile as assayed by RP-HPLC.
Results are shown in FIG. 8. Similarly, the stability of liquid and
gelled (by agitation) aqueous leuprolide formulations (370 mg/ml)
was studied in vitro at 37.degree. C. and in vivo in rats,
respectively. Results are presented in Table II, and show that the
both gelled and liquid formulations remained stable over a period
of 18 weeks.
TABLE II ______________________________________ Stability Studies
of Liquid and Gelled Aqueous Leuprolide Formulations TIME LIQUID
GELLED STUDY (weeks) (% remaining) (% remaining)
______________________________________ Long Term Stab 6 98.00 Long
Term Stab 12 91.50 Long Term Stab 18 93.50 Rat 4 94.80 Rat 6 93.50
Rat 12 92.30 Rat 18 92.60
______________________________________
A major aspect of the invention is that aqueous solutions
containing high concentrations of peptide compounds are stable at
high temperatures for long periods of time. Thus, these
formulations are advantageous in that they may be shipped and/or
stored for long periods of time at or above room temperature. They
are also suitable for use in implantable delivery devices.
DISCLOSURE OF EXAMPLES OF THE INVENTION
The following methods were used to perform the studies in the
Examples that follow.
1. Preparing Leuprolide Acetate Solutions
Leuprolide acetate (obtained, for example, from Mallinckrodt, St.
Louis, Mo.) was weighed, added to a weighed amount of vehicle
(sterile distilled water, ethanol/water or water with non-ionic
surfactant) at the appropriate concentration (w/w), then gently
stirred to dissolve.
Unless otherwise noted, leuprolide free base content was calculated
from certificate of analysis potency values to be 37% free base.
This was 40% leuprolide acetate, except as noted.
2. Preparation of Reservoirs
The reservoirs of implantable drug delivery devices (as disclosed
in U.S. patent application Ser. No. 08/595,761, incorporated herein
by reference) were filled with the appropriate leuprolide acetate
solution. The filled devices then underwent stability testing. The
formulation was filled into titanium or polymer reservoirs with a
polymer plug blocking each end. The filled reservoir was then
sealed in a polyfoil bag and placed in a stability testing
oven.
It should be noted that the formulations in the reservoirs of these
devices are completely isolated from the outside environment.
3. Reverse Phase-HPLC (RP-HPLC)
All stability samples were analyzed for leuprolide concentration
and % peak area using a gradient elution reversed-phase HPLC assay
with a refrigerated autosampler (4.degree. C.) to minimize sample
degradation. The chromatographic conditions used are listed
below.
______________________________________ RP-HPLC Chromatographic
Conditions Description Parameter
______________________________________ Column HaiSil C18, 4.6
.times. 250 mm, S/N 5103051 Flow Rate 0.8 mL min.sup.-1 Injection
Volume 20 .mu.L Detection 210 nm Leuprolide Between 25-30 minutes
Retention Time Mobile Phase A = 100 mM Sodium Phosphate, pH 3.0 B =
90% Acetonitrile/Water Gradient Minutes 0 5 25 40 41 46 46.1 50 %B
15 26.5 26.5 65 85 85 15 15
______________________________________
Leuprolide standards (in water) at 4 to 6 different concentration
levels, typically between 0.1-1.2 mg/mL, were run along with the
stability samples. The stability samples were bracketed by the
standard sets, with no more than 40 samples in between the standard
sets. All peaks between the void volume and 45 minutes of the run
were integrated. The integrated peak areas for the leuprolide
standards were plotted as a function of the concentration. The
leuprolide concentrations for the stability samples were then
calculated using linear regression. The % peak areas for the
leuprolide peak, the sum of all the peaks eluting before
leuprolide
(labeled "others"), and the sum of all the peaks eluting after
leuprolide (labeled "aggregates") were also recorded and plotted as
a function of the sample timepoints.
4. Size Exclusion Chromatography (SEC)
Selected stability samples were analyzed for % peak area and
molecular weights using an isocratic solution SEC assay with a
refrigerated autosampler (4.degree. C.). The chromatographic
conditions used are listed below.
______________________________________ SEC Chromatographic
Conditions Description Parameter
______________________________________ Column Pharmacia Peptide, HR
10/30, 10 .times. 300 mm Flow Rate 0.5 mL min.sup.-1 Injection
Volume 20 .mu.L Detection 210 nm Leuprolide Retention Approximately
25 minutes Time Mobile Phase 100 mM Ammonium Phosphate, pH 2.0, 200
mM Sodium Chloride, 30% Acetonitrile
______________________________________
The void volume and total volume for the size exclusion column was
needed for the calculation of the molecular weights. The BioRad
high molecular weight standard and 0.1% acetone were used to
determine the void volume and total volume respectively. The
retention times for the first peak in the BioRad standard and the
acetone peak were recorded and converted to volume units using the
equations below. Since these values are constant for a particular
SEC column and HPLC system, the void and total volumes were
redetermined whenever changes to the SEC column or HPLC system were
made. A standard run was then made followed by the stability
samples. The standard mixture contained approximately 0.2 mg/mL of
the following peptides: Bursin (MW=449), WLFR peptide (MW=619),
Angiotensin (MW=1181), GRF (MW=5108), and Cytochrome C (MW=12394).
These standards were chosen because they bracketed leuprolide
molecular weight and all had basic pl (9.8-11.0), similar to
leuprolide.
The % peak areas were recorded for all the peaks. The molecular
weights for the species separated were calculated using the
equations below.
V.sub.s =flow rate (mL/min).times.sample peak retention time
(min)
V.sub.o =flow rate (mL/min).times.void volume peak retention time
(min)
V.sub.t =flow rate (mL/min).times.total volume peak retention time
(min) ##EQU1##
Where:
V.sub.s =standard or sample volume
V.sub.o =void volume
V.sub.t =total volume
V.sub.s was calculated to each peptide standard peak. Kd for each
peptide standard was then calculated using the values for V.sub.t
and V.sub.o determined earlier. The linear regression line from the
plot of logMW vs. Kd.sup.-1 was used to determine the molecular
weights for each peak in the stability sample. The % peak areas for
the stability samples were also recorded.
5. Instrumentation and Materials
The instrumentation and materials used for RP-HPLC and SEC were as
follows:
Waters Millennium HPLC system consisting of 717 autosampler, 626
pump, 6000S controller, 900 photodiode array detector, and 414
refractive index detector (Waters Chromatography, Milford,
Mass.)
HPLC vials, for 48-position and 96-position (Waters Chromatography,
Milford, Mass.)
HaiSil C18, 120 A, 5 .mu.m4.6.times.250 mm HPLC column (Higgins
Analytical, Mountain View, Calif.)
Pharmacia Peptide, HR 10/30 SEC column (Pharmacia Biotech,
Piscataway, N.J.)
6. Purity
Stability samples were analyzed using RP-HPLC. The area under the
curve for the leuprolide peak divided by the sum of the areas under
the curve of all peaks gave % purity. [It should be noted that the
data for % concentration presented with the % purity data (Examples
5, 6 and 7) are inconclusive. The analysis methods used to
determine % concentration in these experiments were
unreliable.]
The following examples are offered to illustrate this invention and
are not meant to be construed in any way as limiting the scope of
this invention.
EXAMPLE 1
Accelerated Stability Studies of Leuprolide Acetate
Formulations
Formulations of 40% (w/w) leuprolide acetate (equivalent to about
37% leuprolide free base) in either sterile distilled water,
ethanol/water (70/30) or water with 10% Tween 20 were prepared as
described above and used to fill the reservoirs of implantable drug
delivery devices, also as described above. Some reservoirs were
made of polymer materials, while some were titanium.
The filled devices were subjected to accelerated aging by storing
them at elevated temperatures (80-88.degree. C.) for seven days in
an incubator (Precision Scientific or Thelco). This is equivalent
to about 1.5 years at 37.degree. C. or about four years at room
temperature (25.degree. C.), assuming an activation energy
(E.sub.a) or 22.2 kcal/mole.
The samples were analyzed using RP-HPLC and SEC as described above
to determine the chemical and physical stability of the aged
formulations.
Results, presented in Table III, demonstrate that these aqueous
formulations were able to maintain the stability of the
LHRH-related compound leuprolide. In each case, at least 65%
leuprolide was retained. However, a large amount of the formulation
with EtOH evaporated from the reservoir during the study,
indicating that long term storage at elevated temperatures of
formulations with high concentrations of a volatile solvent like
EtOH may be problematic. The formulation which contained the
non-ionic surfactant 10% Tween 20 was found to be no more stable
that water solutions without this solubilizer.
TABLE III ______________________________________ Stability of 40%
(w/w) Leuprolide Acetate Aqueous Formulations After 7 Days at
Elevated Temperatures Temperature Reservoir % Leuprolide (.degree.
C.) Material Formulation at Day 7
______________________________________ 88 Polymer 40% in Water 68
88 Titanium 40% in Water 71 88 Polymer 40% in Water 66* 88 Polymer
40% in EtOH/H.sub.2 O 85** (07/30) 88 Polymer 40% in 10% Tween 20
65 80 Polymer 40% in Water 83 80 Polymer 40% in Water 80 80 Polymer
40% in Water 78 80 Polymer 40% in Water 79 80 Polymer 40% in Water
83 80 Polymer 40% in Water 77 80 Polymer 40% in Water 79 80 Polymer
40% in Water 74 80 Polymer 40% in Water 88
______________________________________ *10% evaporated **60%
evaporated
EXAMPLE 2
Stability Studies of Irradiated Leuprolide Acetate Formulations
Formulations of 40% (w/w) as received leuprolide acetate
(equivalent to 37% leuprolide free base) in water were prepared as
described above and used to fill the reservoirs of drug delivery
devices, also as described above. Some reservoirs were made of
polymer materials, while some were titanium.
The filled devices were subjected to 2.5 megarad gamma irradiation.
Samples were shipped to Sterigenics (Tustin, Calif.) and gamma
irradiated (Cobalt 60) in batch mode. Samples were then subjected
to accelerated aging as in Example 1. Samples labeled "cold" were
shipped and irradiated on dry ice. Samples were taken at day 0 and
day 7, and analyzed using RP-HPLC and SEC as described above to
determine the chemical and physical stability of the irradiated
formulations.
Results, presented in Table IV, demonstrate that these leuprolide
acetate formulations were stable after irradiation. In every case,
at least 65% leuprolide was retained, with low levels of aggregate
formation.
TABLE IV
__________________________________________________________________________
Stability of 40% (w/w) Leuprolide Acetate Aqueous Formulations
After 2.5 Megarad Gamma Irradiation SEC Day 0 Day 7 Reservoir
Material Formulation Irradiation % Leuprolide at Day 7 (RP-HPLC) %
monomer % dimer/trimer % monomer %
__________________________________________________________________________
dimer/trimer Polymer 40% in Water Yes 75 90.4 1.2 80.9 3.9 Polymer
40% in Water No 75 99.8 0.2 82.4 3.1 Polymer 40% in Water Cold 79
89.4 0.2 80.3 3.1 Titanium 40% in Water Yes 83 98.5 1.1 84.9 2
Titanium 40% in Water No N.D. 99.6 0 96.6 0 Titanium 40% in Water
Yes 81 98.8 0.9 94.7 2.4 Titanium 40% in Water No 82 99.9 0 95 1.9
Titanium 40% in Water Yes 73 99.1 0.9 88.3 3 Titanium 40% in Water
Yes 79 99 0.8 94.3 3.4 Titanium 40% in Water Yes 74 98.6 0.5 90.9
3.6
__________________________________________________________________________
EXAMPLE 3
Long Term Accelerated Stability Studies of Leuprolide Acetate in
Water
Solutions of 40% leuprolide acetate (w/w) in water were prepared,
loaded into reservoirs, stored for two months at 80.degree. C. and
analyzed as described above. Results, shown in FIGS. 1 (RP-HPLC)
and 2 (SEC) show that 81.1% leuprolide was recovered, with only
14.6% chemical degradation and 5.1 % physical aggregation after the
two month period.
Leuprolide acetate solutions (40% (w/w) in water) were prepared,
loaded, stored and analyzed as set forth above. FIG. 4 is a plot of
leuprolide, and its chemical and physical degradation products
recovered over a three month time period. The sum of these three
elements is also presented as mass balance. The results show that
we can account for all the peptide material as either intact
leuprolide or a degradation species, indicating that stability
studies are not missing an unknown degradation process or
product.
Leuprolide acetate solutions (40% (w/w) in water) were prepared,
loaded, stored at 37.degree. C., 50.degree. C., 65.degree. C. or
80.degree. C. and analyzed using RP-HPLC as described above. FIG. 5
shows the loss of leuprolide from these solutions over a three to
six month period, and indicates that leuprolide degradation fits
pseudo first order kinetics. Furthermore, as discussed below, FIG.
3 indicates that leuprolide in water degradation fits linear
Arrhenius kinetics. Therefore, accelerated stability studies are a
valid technique for assessing the stability of leuprolide and
extrapolating back to 37.degree. C.
Solutions of 40% leuprolide acetate (w/w) in water were prepared,
loaded into reservoirs, stored at 37.degree. C., 50.degree. C.,
65.degree. C. or 80.degree. C. and analyzed using RP-HPLC as
described above. Results were calculated as described in Physical
Pharmacy: Physical Chemical Principles in the Pharmaceutical
Sciences, 3rd ed., Martin et al., Chapter 14 (1983) and showed the
E.sub.a of these solutions to be 22.2 kcal/mole with a t.sub.90 of
13.8 months.
The data are shown below and an Arrhenius plot of the data is
presented in FIG. 3.
______________________________________ Water .degree.C. Kobs
(months.sup.-1) t.sub.1/2 (months)
______________________________________ 37 7.24 .times. 10.sup.-3
95.7 50 3.21 .times. 10.sup.-2 21.6 65 0.111 6.3 80 0.655 1.1
______________________________________ E.sub.a = 22.2 kcal/mole
EXAMPLE 4
Long Term Stability Studies of Leuprolide Acetate in Water
The chemical stability of 40% leuprolide acetate solutions prepared
and analyzed as described above is presented in FIG. 6. After nine
months at 37.degree. C.. more than 85% (88.3%) leuprolide was
present, with less than 10% (8.4%) chemical degradation products
(shown as "early" in the figure, based on the RP-HPLC profile) and
less than 5% (3.5%) physical aggregates (shown as "late" in the
figure, based on RP-HPLC data, but in good agreement with SEC
data).
EXAMPLE 5
Accelerated Stability Studies of Goserelin
Formulations of 30% goserelin (w/w) in acetate buffer (pH 5.0,
0.0282M) with 3% mannitol were stored in glass ampules for 14 days
at 80.degree. C. and analyzed for purity as described above.
Results in FIG. 7 show that after 9 days about 65% goserelin
remained.
EXAMPLE 6
Stability Studies of Goserelin Formulations
Formulations of 40-45% (w/w) goserelin in either acetate buffer,
with 3% mannitol or acetate buffer with salt (0.9% NaCl) were
prepared as described above and placed in polymeric containers.
The containers were stored at 37.degree. C. for one month in an
incubator.
The samples were analyzed using RP-HPLC to determine the chemical
stability of the aged formulations.
Results, presented below, demonstrate that these aqueous
formulations were able to maintain the stability of the
LHRH-related compound goserelin. In
each case, at least 98% goserelin was retained.
______________________________________ % CON- DRUG VEHICLE % PURITY
CENTRATION ______________________________________ Goserelin Acetate
Buffer/Mannitol 98.1 54.2 Goserelin Acetate Buffer/Salt 98.0 50.1
______________________________________
EXAMPLE 7
Stability Studies of Nafarelin Formulations
Formulations of 15% (w/w) nafarelin in acetate buffer with 3%
mannitol were prepared as described above and placed in polymeric
containers.
The containers were stored at 37.degree. C. for one month in an
incubator.
The samples were analyzed using RP-HPLC to determine the chemical
stability of the aged formulations.
Results, presented below, demonstrate that these aqueous
formulations were able to maintain the stability of the
LHRH-related compound nafarelin, as at least 98% nafarelin was
retained.
______________________________________ % CON- DRUG VEHICLE % PURITY
CENTRATION ______________________________________ Nafarelin Acetate
Buffer/Mannitol 98.8 18.3
______________________________________
Modification of the above-described modes of carrying out various
embodiments of this invention will be apparent to those of skill in
the art following the teachings of this invention as set forth
herein. The examples described above are not limiting, but are
merely exemplary of this invention, the scope of which is defined
by the following claims.
* * * * *